Recently we have shown that purified vaccinia virus will synthesize a class of high-molecular-weight virion-associated RNAs in vitro. A number of preliminary experiments suggest that a possible role for this high-molecular-weight virion-associated RNA is that of a precursor to virion-extruded 8-12S mRNA. The proposed research will utilize this in vitro viral system as a model for mRNA processing and modification. The proposal will deal with the following: (a) Purification and characterization of the endoribonuclease involved in the cleavage of the high-molecular-weight RNA (approximately 26S) to 8-12S mRNA; (b) Analysis of the methylated residues found on the high-molecular-weight RNA and their fate upon processing; (c) Analysis of the posttranscriptional polyadenylation of fragments cleaved from purified high-molecular-weight virion-associated RNA. The above (a-c) will utilize soluble crude extracts of detergent disrupted viral cores which are known to contain the pertinent enzymes and the purified enzymatic components derived from these extracts; (d) Analysis of the informational content of both precursor and its RNA cleavage products, as defined by their relative ability to be translated into proteins by in vitro systems; and (e) investigation of these events in vaccinia-infected HeLa cells in order to gain an insight into the biological significance of the observations made with purified virions in vitro.